Formulation, Characterization, and Potential Therapeutic Implications of Encapsulated Recombinant Alpha-luffin in Niosomes.

Deregulation of micro-RNAs (miRNAs) is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. To explore the potential of exogenously applied miRNAs to suppress oncogenic proteins, the ERBB oncogene family was chosen with a bioinformatics search identifying targeting seed sequences for miR-125a and miR-125b within the 3'-untranslated regions of both ERBB2 and ERBB3. Using the human breast cancer cell line SKBR3 as a model for ERBB2 and ERBB3 dependence, infection of these cells with retroviral constructs expressing either miR-125a or miR-125b resulted in suppression of ERBB2 and ERBB3 at both the transcript and protein level. Luciferase constructs containing the 3' 3'-untranslated regions of ERBB2 and ERBB3 demonstrated approximately 35% less activity in miR-125a- and miR-125b-expressing cells relative to controls. Additionally, phosphorylation of ERK1/2 and AKT was suppressed in SKBR3 cells overexpressing either miR-125a or miR-125b. Consistent with suppression of both ERBB2 and ERBB3 signaling, miR-125a-or miR-125b-overexpressing SKBR3 cells were impaired in their anchorage-dependent growth and exhibited reduced migration and invasion capacities. Parallel studies performed on MCF10A cells demonstrated that miR-125a or miR-125b overexpression produced only marginal influences on the growth and migration of these non-transformed human mammary epithelial cells. These results illustrate the feasibility of using miRNAs as a therapeutic strategy to suppress oncogene expression and function.

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK.S100A14,one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens,andthere is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding.Moreover,we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.

Differentially expressed proteins among cancer cell lines fit each cell line as a model for gaining knowledge of heterogeneity in cancer. The aim of our study was to identify differentially expressed proteins between MCF7 (ER+, low HER2 expression) and SKBR3 (ER-, high HER2 expression) cell lines by a proteomic approach. Moreover, a number of proteins in MCF7 cell line were randomly selected and identified. MCF7 and SKBR3 cell lysates were subjected to two dimensional gel electrophoresis and spots of interest were identified by MALDI-TOF/TOF mass spectrometry. Upregulated proteins (≥2 fold and p value <0.05) in MCF7 cells were cellular retinoic acid binding-protein 2, Hsp27, nucleophosmin, electron transfer flavoprotein ≤ subunit, and profilin, and in SKBR3 cells were Rho GDP dissociation inhibitor-α (RhoGDI-α), voltage-dependent anion channel 2, aldehyde dehydrogeanase-2 (ALDH2), LDH- A, LDH-B, pyrophosphates 1, GAPDH, cathepsin D preprotein, and apolipoprotein A-I binding. Most of the identified proteins have been a candidate marker for cancer aggressiveness or drug resistance, but their differential expressions between SKBr3 and MCF7 cells were not known. Apo-lipoprotein binding-protein has not been described in cancer so far. Differential expression of RhoGDI-α was further validated by using western blotting with specific antibody. Further studies are required to clarify the importance of differential expressions of the identified proteins in SKBr3 and MCF7 breast cancer cell line models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4785. doi:1538-7445.AM2012-4785

Identifying novel molecular drug targets continues to be of prime interest in addressing the public health burden of breast cancer in both developed and developing countries alike. In this context, proteomics/pharmacoproteomics approaches offer a new dimension for personalized medicine. We have previously identified differentially expressed proteins with antigenic activity between SKBR3 (ER-, high HER2 expression) and MCF7 (ER+, low HER2 expression) breast cancer cell lines. The aims of the present study were (1) to develop an initial proteome based roadmap of differentially expressed proteins between the two cell lines using two-dimensional electrophoresis (2-DE), and (2) to compare them to those identified by other techniques. SKBR3 and MCF7 cell lysates were subjected to 2-DE and spots of interest were identified by MALDI-TOF/TOF MS. Upregulated proteins (≥2 fold and p<0.05) in MCF7 cells were cellular retinoic acid binding-protein-2, Hsp27, nucleophosmin, electron transfer flavoprotein-α, and profilin-2. In SKBR3 cells, upregulated proteins were RhoGDP dissociation inhibitor-α (RhoGDI-α), voltage-dependent anion channel-2, aldehyde dehydrogeanase-2 (ALDH2), LDH-A, LDH-B, pyrophosphates-1, GAPDH, cathepsin-D preprotein, F–actin capping protein β-subunit, and apolipoprotein A-I binding protein. Differential expression of RhoGDI-α, a molecule with a versatile range of biological activities in different types of breast cancer, was validated using western blotting. In conclusion, these observations using proteomics strategies serve to characterize SKBR3 and MCF7 breast cancer cell lines and offer new insights for personalized medicine on differential expression of putative drug targets between these cancer models. Further studies are warranted to examine the usefulness of SKBR3 cell line as an appropriate model for studying RhoGDI-α activities in HER2+ ER- breast cancer. Finally, we underscore that the findings presented herein also attest to an emerging strand of collaborative proteomics/OMICS studies in developing countries and resource-limited settings towards global personalized medicine, an area of postgenomics data-intensive health research that is in need of greater attention in biomedical literature. Keywords: Breast cancer, developing world OMICS, ER, HER2, personalized medicine and developing countries, pharmacoproteomics, proteomics, RhoGDP dissociation inhibitor-α

Introduction: Trastuzumab (T) is a monoclonal antibody therapy used in the treatment of HER2+ breast cancer. T inhibits HER2 intracellular signalling and is capable of engaging the immune system through ADCC. Adenosine is an important negative regulator of the immune response through its interaction with the A2A receptor (A2AR, ADORA2A). Relieving adenosine-mediated immunosuppression by inhibiting A2AR may improve NK cell-mediated T-ADCC against HER2+ breast cancer cells. In addition, we have previously shown that SKBR3 cells resistant to the EGFR/HER2 tyrosine kinase inhibitor (TKI) lapatinib are less sensitive to T-ADCC and showed increased A2AR protein levels. This study examines the effects of inhibiting A2AR signalling on NK cell-mediated T-ADCC against treatment naïve HER2+ breast cancer cell lines HCC1954 and SKBR3 and lapatinib and afatinib (irreversible pan-HER-family TKI)-resistant sublines of HCC1954 and SKBR3. Methods: HER2+ breast cancer cell lines SKBR3 and HCC1954 were exposed to afatinib (150nM) or lapatinib (1μM) for 6 months to generate TKI-resistant SKBR3-A and HCC1954-L cell lines. Acid-phosphatase-based proliferation assays were used to confirm resistance to TKI treatment. Western blotting was used to examine A2AR and HER2 protein levels in cell lines. NK cells were isolated from healthy volunteer whole blood by MACSxpress isolation kits. Immune cell-mediated cytotoxicity was determined at a 1:1 (NK cell: TC) ratio over 12 hours using a flow cytometry-based method. Direct cytotoxicity and T-ADCC were determined +/- A2AR agonist CGS21680 (1 μM) and/or A2AR antagonist preladenant (100 nM) for all cell lines. Experiments were carried out three times with three separate volunteer samples with representative results presented. Results: HCC1954-L cells were 5.3-fold resistance to lapatinib (IC50 1.65 μM +/- 0.22 μM) vs. HCC1954 (IC50 0.31 μM +/- 0.15 μM). SKBR3-A cells were 33-fold resistant to afatinib (IC50 0.28 μM +/- 0.006 nM) vs. the parental SKBR3 cell line (IC50 0.009 μM +/- 0.006 μM). SKBR3 and HCC1954 expressed detectable protein levels of A2AR. A2AR and HER2 levels were not significantly changed between parental and resistant cell lines. Levels of direct cytotoxicity and T-ADCC elicited by NK cells were higher against SKBR3-A (p=0.002) and HCC1954-L cells (p=0.0004) than parental cell lines. The A2AR agonist CGS21680 alone had inconsistent effects on direct cytotoxicity and T-ADCC in all cell lines tested. The addition of A2AR antagonist preladenant to CGS21680, but not preladenant alone, increased T-ADCC against the parental HCC1954 cells by 12.7 +/- 3.4% and parental SKBR3 cells by 9.5 +/- 3.6%. T-ADCC levels in the targeted therapy-resistant HCC1954-L and SKBR3-A cell lines were not impacted by the CGS21680/preladenant combination. Conclusions: A HER2-targeted therapy resistance phenotype is associated with increased T-ADCC in the models tested. Inhibition of activated A2AR can increase T-ADCC elicited by NK cells against treatment naïve HER2+ breast cancer cell lines but not TKI-resistant sublines. Further work is warranted to examine the impact of targeting A2AR in HER2+ breast cancer. Citation Format: Gaynor N, Noone J, Monedero J, Murphy EE, O'Gorman DJ, Crown J, Collins DM. The effect of relieving adenosine-mediated immunosuppression on trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity (T-ADCC) against HER2+ breast cancer cell lines [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-06-19.

The advent of pH-sensitive liposomes (pHLips) has opened new opportunities for the improved and targeted delivery of antitumor drugs as well as gene therapeutics. Comprising fusogenic dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS), these nanosystems harness the acidification in the tumor microenvironment and endosomes to deliver drugs effectively. pH-responsive liposomes that are internalized through endocytosis encounter mildly acidic pH in the endosomes and thereafter fuse or destabilize the endosomal membrane, leading to subsequent cargo release into the cytoplasm. The extracellular tumor matrix also presents a slightly acidic environment that can lead to the enhanced drug release and improved targeting capabilities of the nano-delivery system. Recent studies have shown that folic acid (FA) and iRGD-coated nanocarriers, including pH-sensitive liposomes, can preferentially accumulate and deliver drugs to breast tumors that overexpress folate receptors and αvβ3 and αvβ5 integrins. This study focuses on the development and characterization of 5-Fluorouracil (5-FU)-loaded FA and iRGD surface-modified pHLips (FA-iRGD-5-FU-pHLips). The novelty of this research lies in the dual targeting mechanism utilizing FA and iRGD peptides, combined with the pH-sensitive properties of the liposomes, to enhance selective targeting and uptake by cancer cells and effective drug release in the acidic tumor environment. The prepared liposomes were small, with an average diameter of 152 ± 3.27 nm, uniform, and unilamellar, demonstrating efficient 5-FU encapsulation (93.1 ± 2.58%). Despite surface functionalization, the liposomes maintained their pH sensitivity and a neutral zeta potential, which also conferred stability and reduced aggregation. Effective pH responsiveness was demonstrated by the observation of enhanced drug release at pH 5.5 compared to physiological pH 7.4. (84.47% versus 46.41% release at pH 5.5 versus pH 7.4, respectively, in 72 h). The formulations exhibited stability for six months and were stable when subjected to simulated biological settings. Blood compatibility and cytotoxicity studies on MDA-MB-231 and SK-BR3 breast cancer cell lines revealed an enhanced cytotoxicity of the liposomal formulation that was modified with FA and iRGD compared to free 5-FU and minimal hemolysis. Collectively, these findings support the potential of FA and iRGD surface-camouflaged, pH-sensitive liposomes as a promising drug delivery strategy for breast cancer treatment.

This study aims to evaluate Succinate Dehydrogenase Complex Flavoprotein Subunit A (SDHA) expression across various breast cancer subtypes, its prognostic significance, and the impact of SDHA knockdown on breast cancer cell functions. To assess SDHA expression in breast cancer, we utilized multiple publicly available databases. Prognostic significance was also evaluated using relevant databases. Methylation status, and enrichment analysis were performed using the GSCA database. The mutational status of SDHA was examined using cBioPortal, and its relationship with immune infiltration and drug sensitivity was assessed. Functional assays, including cell proliferation, colony formation, wound healing, and SDHA knockdown, were performed using MCF-7 and SKBR3 breast cancer cell lines. Our results showed that SDHA was significantly overexpressed in breast cancer tissues compared to normal tissues. High SDHA expression was correlated with worse survival in breast cancer patients. Pathological stage analysis revealed that SDHA expression increased as the disease progressed, with lower methylation levels in tumor tissues suggesting epigenetic regulation of its expression. Functionally, SDHA knockdown in MCF-7 and SKBR3 cells led to significant reductions in cell proliferation, colony formation, and migration, highlighting its role in supporting breast cancer cell growth and metastasis. SDHA was upregulated in breast cancer and associated with poor prognosis. Our findings also suggest that SDHA plays a crucial role in promoting breast cancer cell growth and migration, indicating its therapeutic potential. Targeting SDHA could provide a novel strategy for breast cancer treatment, particularly in overcoming chemoresistance and inhibiting tumor progression.

We analyzed whether polypurine hairpins (PPRHs) had the ability to knock down gene expression. These hairpins are formed by two antiparallel purine domains linked by a loop that allows the formation of Hoogsteen bonds between both domains and Watson-Crick bonds with the target polypyrimidine sequence, forming triplex structures. To set up the experimental conditions, the human dhfr gene was used as a model. The PPRHs were designed toward the template strand of DNA. The transfection of the human breast cancer cell line SKBR3 with these template hairpins against the dhfr gene produced higher than 90% of cell mortality. Template PPRHs produced a decrease in DHFR mRNA, protein, and its corresponding enzymatic activity. In addition, the activity of DHFR PPRHs was tested against breast cancer cells resistant to methotrexate, observing high cell mortality. Given the difficulty in finding long polypyrimidine stretches, we studied how to compensate for the presence of purine interruptions in the polypyrimidine target sequence. The stability of PPRH was measured, resulting in a surprisingly long half-life of about 5 days. Finally, to test the generality of usage, template PPRHs were employed against two important genes involved in cell proliferation, telomerase and survivin, producing 80 and 95% of cell death, respectively. Taken together our results show the ability of antiparallel purine hairpins to bind the template strand of double strand DNA and to decrease gene transcription. Thus, PPRHs can be considered as a new type of molecules to modulate gene expression.

Co-amplification and co-overexpression of ErbB2 and Grb7 are frequently found in various cancers, including breast cancer. Biochemical and functional correlations of the two molecules have identified Grb7 to be a pivotal mediator downstream of ErbB2-mediated oncogenesis. However, it remains largely unknown how Grb7 is involve in the ErbB2-mediated tumorigenesis. In this study, we show that Grb7-mediated cell proliferation and growth are essential for the tumorigenesis that occurs in ErbB2-Grb7-overexpressing breast cancer cells. Intrinsically, EGF-induced de novo Grb7 tyrosine phosphorylation/activation recruits and activates Ras-GTPases and subsequently promotes the phosphorylation of ERK1/2, thereby stimulating tumor growth. Furthermore, we also found the anti-tumor effect could be synergized by co-treatment with Herceptin plus Grb7 knockdown in Sk-Br3 breast cancer cells. Our findings illustrate an underlying mechanism by which Grb7 promotes tumorigenesis through the formation of a novel EGFR-Grb7-Ras signaling complex, thereby highlighting the potential strategy of targeting Grb7 as an anti-breast cancer therapy.

Abstract #1039 Introduction: The HER family of receptors HER1 (EGFR/ErbB1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4) is critical in the regulation of cell growth, proliferation and survival. Several studies have linked over-expression of HER1 and HER2 with resistance to chemotherapy and radiation. Clinical trials combining the HER tyrosine kinase inhibitors (TKI) gefitinib (GEF), an inhibitor of HER1, and lapatinib (LAP), a dual inhibitor of HER1 and HER2, with chemotherapeutic agents have not shown synergy comparable to that found in preclinical models. We investigated the effects of chronic treatment of breast cancer cells with TKI inhibitors on DNA damage and repair resulting from chemotherapy and ionising radiation (IR).&amp;#x2028; Material and Methods: Western blotting was used to examine the expression of phosphorylated HER receptors, AKT and MAPK in the SKBR3 and BT474 human breast cancer cell lines. Etoposide (ET), doxorubicin (DOX) and IR induced DNA strand breaks (SB) were measured in the SKBR3 cell line using the single cell gel electrophoresis (comet) assay. Cisplatin-induced (PT) interstrand crosslinks (ICL) were measured with the modified alkaline comet assay. Cells were pre-treated with LAP or GEF for 1 hour or 48 hours, and TKI was replaced at 24-hourly intervals. Cell cycle analysis and intracellular DOX were measured by flow cytometry.&amp;#x2028; Results: In the SKBR3 and BT474 cell lines pHER1, pHER2, pHER3, pAKT and pMAPK were inhibited within 1 hour of exposure to LAP or GEF. However, after 48 hours, pHER3 and pAKT signalling was detected in cells treated with GEF, but not LAP. pHER1, pHER2 and pMAPK were inhibited for at least 72 hours with both drugs. Cell cycle analysis on the SKBR3 cell line showed that 48 hours exposure to either TKI reduced the number of cells in S (12.4±1.1% to 3.2±0.3%), and G2 (16.0±0.55% to 7.1±1.0%) phases of the cell cycle. There was no alteration in intracellular DOX levels at 1 hour or following 48 hours exposure to either TKI. SB were induced by ET, DOX and IR, and ICL by PT, in cells exposed to GEF or LAP for 1 hour. Exposure for 48 hours did not alter the number of ICL or SB induced by PT or IR. In contrast there was a 30-90% reduction in SB following ET treatment (depending on drug concentration) in cells treated with TKI for 48 hours compared with 1 hour treatment. The effect of 48 hours exposure to TKI on DOX-induced SB was more marked with &amp;gt;90% reduction, even at high DOX concentrations. Higher concentrations of ET resulted in increased SB, but increasing concentration of DOX did not alter the level of detectable SB.&amp;#x2028; Discussion: These results suggest why clinical trials of combinations of HER TKI with chemotherapy agents fail to show the magnitude of synergy predicted in preclinical studies. Whilst the cytostatic properties of both LAP and GEF may explain the reduction in DNA damage after 48 hours, this does explain why SB are absent in DOX-treated cells. Further studies investigating kinetics of DNA repair following chemotherapy or radiation in combination with TKI are on-going. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1039.

BackgroundDuring the past few years, there has been an increasing interest among the Traditional and Folk medical practitioners of Sri Lanka in the use of a decoction prepared from Flueggea leucopyrus (Willd.) for treating various cancers including breast cancer. In the present study, the cytotoxicity of this decoction and its effects on Heat Shock Protein (HSP) expression and apoptosis were compared in three breast cancer phenotypes, to scientifically evaluate if a decoction prepared from F. leucopyrus (Willd.) is useful for the treatment of breast cancer.MethodsCytotoxic potential of the F. leucopyrus decoction was determined by evaluating its effects in MCF-7, MDA-MB-231 and SKBR-3 breast cancer cell lines, and MCF-10A (non-cancerous) breast cell line, by use of the Sulphorhodamine (SRB) assay. The effect of the decoction on HSP gene expression in the above cells was evaluated by (a) Real time reverse transcription PCR (RT-PCR) and (b) Immunofluorescence analysis of HSP protein expression. Effects of the decoction on apoptosis were evaluated by (a) fluorescent microscopic examination of apoptosis related morphological changes and (b) DNA fragmentation (c) Caspase 3/7 assay.ResultsF. leucopyrus decoction can mediate significant cytotoxic effects in all three breast cancer cells phenotypes (IC50 values: 27.89, 99.43, 121.43 μg/mL at 24 h post incubation periods, for MCF-7, MDA-MB-231, SKBR-3 respectively) with little effect in the non-cancerous breast cell line MCF-10A (IC50: 570.4 μg/mL). Significant (*P <0.05) inhibitions of HSP 90 and HSP 70 expression were mediated by the decoction in MCF-7 and MDA-MB-231, with little effect in the SKBR-3 cells. Clear apoptotic morphological changes on Acridine orange/Ethidium bromide staining and DNA fragmentation were observed in all three breast cancer cell lines. Caspase 3/7 were significantly (*P <0.05) activated only in MDA-MB-231 and SKBR-3 cells indicating caspase dependent apoptosis in these cells and caspase independent apoptosis in MCF-7 cells.ConclusionsModulation of HSP 90 and HSP 70 expressions is a possible mechanism by which the decoction of F. leucopyrus mediates cytotoxic effects MCF-7 and MDA-MB-231 cells. This effect appears to correlate with enhanced apoptosis in these cells. In SKBR-3 cells, mechanisms other than HSP inhibition may be utilized to a greater extent by the decoction to mediate the observed cytotoxic effects. Overall findings suggest that the decoction has the potential to be exploited further for effective treatment of breast cancer.

HER2-positive breast cancer is an aggressive subtype characterized by the overexpression of the HER2 receptor, a transmembrane glycoprotein critical for tumor progression. Current therapies often face challenges like drug resistance and systemic toxicity, necessitating the development of advanced drug delivery systems. This study aimed to fabricate and determine the cytotoxicity of pH-sensitive PLA nanoparticles dual-loaded with docetaxel and each of the small molecule tyrosine kinase inhibitors (STKIs) (tucatinib, neratinib, lapatinib) in HER2-positive breast cancer cells. Nanoparticles were synthesized by a dispersion polymerization method using an acidlabile crosslinking agent, and PEG and lactide macromonomers. They were characterized for structure (TEM), surface morphology (SEM), particle size, polydispersity index, zeta potential, and drug loading capacity. Cytotoxicity was assessed in vitro on SKBR3 and MCF7 breast cancer cell lines, with IC50 values compared across formulations. The nanoparticles were spherical with nanoscale sizes and negative zeta potential values. In vitro studies demonstrated enhanced antiproliferative effects of the drug-loaded nanoparticles, with synergistic activity observed between docetaxel and the STKIs. The drug concentrations were halved in combination formulations and resulted in better cytotoxicity compared to single-drug treatments, particularly against SKBR3 cells. The IC50 values were lower in SKBR3 cells than in MCF7 cells, highlighting the role of HER2 expression in the activity of TKIs. The pH-sensitive PLA nanoparticles effectively co-delivered docetaxel and STKIs and demonstrated enhanced efficacy and reduced drug dosages in HER2-positive breast cancer models. This study provides a foundation for further exploration of nanoparticle-based combination therapies with potential applications in treating other aggressive cancer types.

Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation

IntroductionBreast cancer is among the most prevalent type of cancer in women across the world. The essential oils of Pistacia atlantica Desf. have been utilized as a medicine owing to their antibacterial, antioxidant, anti-inflammatory, and anticancer effects. The current research aims to enhance the anticancer effect of the essential oil of Pistacia atlantica Desf. by incorporating it in a nanostructured lipid carrier (NLC), a lipid-based drug delivery system. MethodsNLCs-containing Pistacia atlantica Desf. essential oil was prepared by probe ultrasonication method to obtain Pistacia atlantica Desf. EO-loaded NLCs (PAEO-NLCs) and its anticancer effect against SKBR3 breast cancer cells was investigated. Dynamic light scattering (DLS) method was used to determine the particle size, as well as polydispersity index (PDI) and zeta potential (ZP) of formulations. Transmission electron microscopy (TEM) was also used to determine the size and morphology of optimized formulation (PAEO-NLC4). In vitro cellular assays (MTT, apoptosis, and cell cycle) were employed to assess the anticancer effect of PAEO-NLC4 against SKBR3 cells. ResultsTEM results revealed that PAEO-NLC4 have a well-defined spherical shape, with a size of 151 ± 1 nm, with a negative surface charge of − 29.1 ± 1.4 mV, and a PDI of 0.16 ± 0.03. Cellular data revealed that the PAEO-NLC4 can decline the viability of SKBR3 cells through cell cycle arrest and apoptosis, compared to cells treated with placebo and free PAEO. ConclusionsBased on our findings, PAEO-NLC4 could serve as a potential and effective treatment for breast cancer therapy.

Purpose: Autophagy is a critical physiological process of cell survival in the presence of stress and is an important cause of resistance to anti-cancer therapy. Drugs that inhibit autophagy (e.g. chloroquine) deprive the cells of this protective mechanism and therefore are promising agents in a combination therapy of breast cancer. Our group has designed and synthesized a novel retinoic acid derivative, VN/12-1. We reported previously that VN/12-1 is a potent inducer of autophagy in estrogen independent, HER2 over-expressing breast cancer cell line SK-BR3 in vitro. In the present study, we used chloroquine in combination with VN/12-1 in an SK-BR3 xenograft model to test the hypothesis that the combination of VN/12-1 and chloroquine would result in greater efficacy against SK-BR3 tumors compared to VN/12-1 alone or all-trans retinoic acid (ATRA) alone. Experimental Design: To test this hypothesis, 64 female SCID mice (8 mice per group) were inoculated with SK-BR3 cells. Once the tumors formed, the mice were treated with vehicle (control), VN/12-1 and ATRA alone or in combination with chloroquine for 30 days. The two dosages of VN/12-1 for the groups involving VN/12-1 were 2.5 mg/kg and 5 mg/kg. The doses of ATRA and chloroquine were 5 mg/kg and 50 mg/kg, respectively. All the agents were administered subcutaneously (s.c) twice a week. Results: Out of all the treatment groups, both the dosages of VN/12-1 (2.5 mg/kg and 5mg/kg) when used in combination with chloroquine were the most effective in inhibiting the growth of SK-BR3 tumors (70% and 80% growth inhibition respectively compared to vehicle control). The combination of ATRA with chloroquine did not show statistically significant inhibition of tumor growth. The effect of each treatment on the body weight and the weights of uteri was also determined. No toxicities were observed as noted by no significant changes in the weight of the mice. The data about the tumor samples’ analysis for the effect of each treatment on the expression of various tumor markers and autophagy markers and pharmacokinetic studies on VN/12-1 will be presented. Conclusion: Our studies have demonstrated for the first time that the combination of VN/12-1 and chloroquine can be used to treat estrogen independent breast cancer. It is hoped that this combination therapy would solve the problem of the development of acquired resistance due to autophagy that typically follows therapeutic treatment of breast cancer. We envision further advanced preclinical development of this combination therapy as potential therapy for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 659. doi:10.1158/1538-7445.AM2011-659